Tyrosine Deprotonation and Associated Hydrogen Bond Rearrangements in a Photosynthetic Reaction Center

Photosynthetic reaction centers from Blastochloris viridis possess Tyr-L162 located mid-way between the special pair chlorophyll (P) and the heme (heme3). While mutation of the tyrosine does not affect the kinetics of electron transfer from heme3 to P, recent time-resolved Laue diffraction studies reported displacement of Tyr-L162 in response to the formation of the photo-oxidized P+•, implying a possible tyrosine deprotonation event. pKa values for Tyr-L162 were calculated using the corresponding crystal structures. Movement of deprotonated Tyr-L162 toward Thr-M185 was observed in P+• formation. It was associated with rearrangement of the H-bond network that proceeds to P via Thr-M185 and His-L168

Overexpression of Protochlorophyllide Oxidoreductase C Regulates Oxidative Stress in Arabidopsis

Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen (1O2). As there is no enzymatic detoxification mechanism available in plants to destroy 1O2, its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the 1O2 generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of 1O2 and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to 1O2-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis

Striated Muscle Regulation of Isometric Tension by Multiple Equilibria

Cooperative activation of striated muscle by calcium is based on the movement of tropomyosin described by the steric blocking theory of muscle contraction. Presently, the Hill model stands alone in reproducing both myosin binding data and a sigmoidal-shaped curve characteristic of calcium activation (Hill TL (1983) Two elementary models for the regulation of skeletal muscle contraction by calcium. Biophys J 44: 383–396.). However, the free myosin is assumed to be fixed by the muscle lattice and the cooperative mechanism is based on calcium-dependent interactions between nearest neighbor tropomyosin subunits, which has yet to be validated. As a result, no comprehensive model has been shown capable of fitting actual tension data from striated muscle. We show how variable free myosin is a selective advantage for activating the muscle and describe a mechanism by which a conformational change in tropomyosin propagates free myosin given constant total myosin. This mechanism requires actin, tropomyosin, and filamentous myosin but is independent of troponin. Hence, it will work equally well with striated, smooth and non-muscle contractile systems. Results of simulations with and without data are consistent with a strand of tropomyosin composed of ∼20 subunits being moved by the concerted action of 3–5 myosin heads, which compares favorably with the predicted length of tropomyosin in the overlap region of thick and thin filaments. We demonstrate that our model fits both equilibrium myosin binding data and steady-state calcium-dependent tension data and show how both the steepness of the response and the sensitivity to calcium can be regulated by the actin-troponin interaction. The model simulates non-cooperative calcium binding both in the presence and absence of strong binding myosin as has been observed. Thus, a comprehensive model based on three well-described interactions with actin, namely, actin-troponin, actin-tropomyosin, and actin-myosin can explain the cooperative calcium activation of striated muscle

Real-Time Decision Fusion for Multimodal Neural Prosthetic Devices

The field of neural prosthetics aims to develop prosthetic limbs with a brain-computer interface (BCI) through which neural activity is decoded into movements. A natural extension of current research is the incorporation of neural activity from multiple modalities to more accurately estimate the user's intent. The challenge remains how to appropriately combine this information in real-time for a neural prosthetic device., i.e., fusing predictions from several single-modality decoders to produce a more accurate device state estimate. We examine two algorithms for continuous variable decision fusion: the Kalman filter and artificial neural networks (ANNs). Using simulated cortical neural spike signals, we implemented several successful individual neural decoding algorithms, and tested the capabilities of each fusion method in the context of decoding 2-dimensional endpoint trajectories of a neural prosthetic arm. Extensively testing these methods on random trajectories, we find that on average both the Kalman filter and ANNs successfully fuse the individual decoder estimates to produce more accurate predictions.Our results reveal that a fusion-based approach has the potential to improve prediction accuracy over individual decoders of varying quality, and we hope that this work will encourage multimodal neural prosthetics experiments in the future

Arp2/3 Branched Actin Network Mediates Filopodia-Like Bundles Formation In Vitro

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent

AbstractThe gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27–35 nm. The analysis of scanning TEM images revealed particles of mass ∼4.3 MDa, representing 101±11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring

Automated identification of multiple micro-organisms from resequencing DNA microarrays

There is an increasing recognition that detailed nucleic acid sequence information will be useful and even required in the diagnosis, treatment and surveillance of many significant pathogens. Because generating detailed information about pathogens leads to significantly larger amounts of data, it is necessary to develop automated analysis methods to reduce analysis time and to standardize identification criteria. This is especially important for multiple pathogen assays designed to reduce assay time and costs. In this paper, we present a successful algorithm for detecting pathogens and reporting the maximum level of detail possible using multi-pathogen resequencing microarrays. The algorithm filters the sequence of base calls from the microarray and finds entries in genetic databases that most closely match. Taxonomic databases are then used to relate these entries to each other so that the microorganism can be identified. Although developed using a resequencing microarray, the approach is applicable to any assay method that produces base call sequence information. The success and continued development of this approach means that a non-expert can now perform unassisted analysis of the results obtained from partial sequence data

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

BACKGROUND:BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to alpha-tubulin and BtubA to beta-tubulin. A 1:1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament. METHODOLOGY/PRINCIPAL FINDINGS:We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1:1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity. CONCLUSIONS:An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsiz

A Tethered Bilayer Assembled on Top of Immobilized Calmodulin to Mimic Cellular Compartmentalization

International audienceBACKGROUND: Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a "trans" side, one to a few nanometer thick, located between the supporting surface and the membrane; and a "cis" side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a "cis" side corresponding to the extracellular milieu and a "trans" side marked by a key cytosolic signaling protein, calmodulin. METHODOLOGY/PRINCIPAL FINDINGS: We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side). CONCLUSIONS: The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules